DNA encoding chimeric toxin

ABSTRACT

A chimeric toxin comprising protein fragments joined together by peptide bonds, the chimeric toxin comprising, in sequential order, beginning at the amino terminal end of the chimeric toxin, 
     (a) the enzymatically active Fragment A of diphtheria toxin, 
     (b) a first fragment including the cleavage domain 1 1  adjacent the Fragment A of diphtheria toxin, 
     (c) a second fragment comprising at least a portion of the hydrophobic transmembrane region of Fragment B of diphtheria toxin, the second fragment having a deletion of at least 50 diphtheria toxin amino acid residues, the deletion being C-terminal to the portion of the transmembrane region, and the second fragment not including domain 1 2 , and 
     (d) a third fragment comprising a portion of a cell-specific polypeptide ligand, the portion including at least a portion of the binding domain of the polypeptide ligand, the portion of the binding domain being effective to cause the chimeric toxin to bind selectively to a predetermined class of cells to be attacked by the enzymatically active Fragment A.

This application is a divisional application of Ser. No. 08/479,107,filed Jun. 7, 1995, now U.S. Pat. No. 5,763,250, which is a continuationof Ser. No. 08/231,397, filed Apr. 22, 1994, now U.S. Pat. No.5,616,482, which is a continuation of Ser. No. 07/886,715, filed May 21,1992, now abandoned, which is a continuation of Ser. No. 07/537,430,filed Jun. 13, 1990, now abandoned, which is a continuation-in-part ofSer. No. 07/488,608, filed Mar. 2, 1990, now abandoned.

BACKGROUND OF THE INVENTION

This invention relates to the use of recombinant DNA techniques toconstruct chimeric toxin molecules.

The literature contains many examples of fused genes which code forchimeric proteins. For example, Villa-Komaroff et al. (1978) Proc. Natl.Acad. Sci. U.S.A. 75:3727-3731, describes a fused gene made up of aeukaryotic structural gene fused to a non-cytoplasmic bacterial gene.The fused gene codes for a chimeric protein which is transported out ofthe cytoplasm. Murphy U.S. Pat. No. 4,675,382, hereby incorporated byreference, describes the use of recombinant DNA techniques to produce ahybrid, or chimeric, protein, consisting of a portion of the diphtheriatoxin (DT) molecule linked via a peptide linkage to a cell-specificligand such as α-melanocyte stimulating hormone (MSH). The DT-MSHchimeric toxin was selectively toxic for particular target cells, i.e.,α-MSH receptor positive human malignant melanoma cells.

A diphtheria toxin-related fusion protein, DAB₄₈₆ -IL-2, in which thenative receptor binding domain of DT was genetically replaced with aportion of the polypeptide hormone interleukin-2 (IL-2) has beendescribed in Williams et al. (1987) Protein Engineering 1:493-498,hereby incorporated by reference. DAB₄₈₆ -IL-2 is a 68,142 Da fusionprotein consisting of, in the following order: Met; DT residues 1-485;and amino acids 2 through 133 of mature human IL-2. DAB₄₈₆ -IL-2 hasbeen shown to bind to the IL-2 receptor and to selectively intoxicatelymphocytes which bear the high affinity form of the IL-2 receptor,Bacha et al. (1988) J. Exp. Med 167:612-622. Moreover, the cytotoxicaction of DAB₄₈₆ -IL-2, like that of native diphtheria toxin, requiresreceptor-mediated endocytosis, passage through an acidic compartment,and delivery of Fragment A associated ADP-ribosyltransferase to thecytosol of target cells, Bacha et al. (1988) supra.

SUMMARY OF THE INVENTION

In general, the invention features a chimeric toxin including proteinfragments joined together by peptide bonds. The chimeric toxin includes,in sequential order, beginning at the amino terminal end of the chimerictoxin:

(a) the enzymatically active Fragment A of diphtheria toxin;

(b) a first fragment including the cleavage domain 1₁ adjacent FragmentA of diphtheria toxin;

(c) a second fragment including at least a portion of the hydrophobictransmembrane region of Fragment B of diphtheria toxin, the secondfragment also having a deletion, C-terminal to the transmembrane region,of at least 50, or more preferably of at least 80, diphtheria toxinamino acid residues, and the second fragment not including domain 1₂ ;and

(d) a third fragment including a portion of a cell-specific polypeptideligand e.g., an interleukin (preferably interleukin 2, or, epidermalgrowth factor (EGF), including at least a portion of the binding domainof the polypeptide ligand, that portion being effective to cause thechimeric toxin to bind selectively to a predetermined class of cells tobe attacked by enzymatically active Fragment A.

In preferred embodiments the chimeric toxin possesses at least one of,and more preferably at least two of, and even more preferably at leastthree of: greater toxicity to receptor-bearing cells than that of ananalagous DAB₄₈₆ -containing-toxin (an analagous DAB₄₈₆ -containingtoxin is a toxin which is identical to the chimeric toxin of thepreferred embodiment except that DAB₄₈₆ replaces the fragments of DTrecited in (a), (b), and (c) above, i.e., a toxin consisting of DAB₄₈₆fused to the fragment defined in (d) above); a lower K_(d) (i.e., agreater binding affinity) for the receptor (i.e., the sites to which thethird fragment (described above) binds on the cells to be attacked) thanthat of an analagous DAB₄₈₆ -containing-toxin; greater resistance toproteolytic degradation than that of DAB₄₈₆ -containing-toxin; greaterresistance to the inhibition of its cytotoxicity by competitiveinhibitors, e.g., the polypeptide of (d) above, than that exhibited byan analagous DAB₄₈₆ -containing-toxin; the ability to inhibit proteinsynthesis in target cells to a given degree by a period of exposure thatis shorter than the period of exposure required by an analogous DAB₄₈₆-containing-toxin to inhibit protein synthesis to the same degree; orthe ability to effect a more rapid onset of the inhibition of proteinsynthesis than that seen in an analagous DAB₄₈₆ -containing-toxin.

Other preferred embodiments include: chimeric toxins wherein thefragment of Fragment B of diphtheria toxin does not include anydiphtheria toxin sequences between the hydrophobic transmembrane regionand amino acid residues 484 or 485 of native diphtheria toxin; chimerictoxins lacking diphtheria toxin sequences C-terminal to amino acidresidue 386 of native diphtheria toxin; and chimeric toxins includingDAB₃₈₉ fused to the third fragment defined above.

Other preferred embodiments include: a chimeric toxin in which theportion of the polypeptide ligand is a portion of interleukin-2effective to cause the chimeric toxin to bind to IL-2 receptor bearingcells, in particular, T cells; a chimeric toxin in which the portion ofthe polypeptide ligand is a portion of EGF effective to cause thechimeric toxin to bind to cells bearing the EGF receptor; the chimerictoxin DAB₃₈₉ -IL-2; and the chimeric toxin DAB₃₈₉ -EGF.

In other preferred embodiments in which the ligand is IL-2 or a portionthereof, the chimeric toxin possesses at least one of: greater toxicityto IL-2 receptor-bearing cells than that exhibited by DAB₄₈₆ -IL-2, alower K_(d) for the IL-2 high affinity receptor than that of DAB₄₈₆-IL-2, or a greater resistance to proteolytic degradation than thatexhibited by DAB₄₈₆ -IL-2.

In other preferred embodiments in which the ligand is EGF or a portionthereof, the chimeric toxin posseses at least one of: greater toxicityto EGF-receptor-bearing cells than that exhibited by DAB₄₈₆ EGF; a lowerK_(d) for the EGF receptor than that of DAB₄₈₆ EGF, greater resistanceto the inhibition of its cytotoxicity by competetive inhibitors, e.g.,EGF, than that of DAB₄₈₆ -EGF; the ability to inhibit protein synthesisin EGF receptor bearing cells to a given degree by a period of exposurethat is shorter than the period of exposure required by DAB₄₈₆ EGF toinhibit protein synthesis to the same degree; or the ability to effect amore rapid onset of the inhibition of protein synthesis inEGF-receptor-bearing cells than that seen in DAB₄₈₆ EGF.

The chimeric toxins of the invention are preferably encoded by fusedgenes which include regions encoding the protein fragments of thechimeric toxin, DNA sequences encoding the chimeric toxins of theinvention, expression vectors encoding those DNA sequences, cellstransformed with those expression vectors, and methods of producing thechimeric toxins including culturing cells transformed with expressionvectors containing DNA encoding the chimeric toxins and isolating thechimeric toxins from the cells or their supernatants.

Native diphtheria toxin, as used herein, means the 535 amino aciddiphtheria toxin protein secreted by Corynebacterium diphtheriae. Thesequence of an allele of the gene which encodes native diphtheria toxincan be found in Greenfield et al. (1983) Proc. Natl. Acad. Sci. USA80:6853-6857, hereby incorporated by reference. Enzymatically activeFragment A, as used herein, means amino acid residues Gly 1 through Arg193 of native DT, or an enzymatically active derivative or analog of thenatural sequence. Cleavage domain 1₁, as used herein, means the proteasesensitive domain within the region spanning Cys 186 and Cys 201 ofnative DT. Fragment B, as used herein, means the region from Ser 194through Ser 535 of native DT. The hydrophobic transmembrane region ofFragment B, as used herein, means the amino acid sequence bearing astructural similarity to the bilayer-spanning helices of integralmembrane proteins and located approximately at or derived from aminoacid residue 346 through amino acid residue 371 of native diphtheriatoxin. Domain 1₂, as used herein, means the region spanning Cys 461 andCys 471 of native DT. The generalized eukaryotic binding site ofFragment B, as used herein, means a region within the C-terminal 50amino acid residues of native DT responsible for binding DT to itsnative receptor on the surface of eukaryotic cells. The chimeric toxinsof the inventions do not include the generalized eukaryotic binding siteof Fragment B.

Toxic or cytotoxic, as used herein, means capable of inhibiting proteinsynthesis in a cell, inhibiting cell growth or division, or killing acell.

DAB₄₈₆ consists of, in the following order, methionine, and amino acidresidues 1-485 of native DT.

DAB₃₈₉ consists of, in the following order, methionine, amino acidresidues 1-386 of native DT, and amino acid residues 484-485 of nativeDT.

DAB₄₈₆ -IL-2 is a fusion protein consisting of, in the following order,methionine, amino acid residues 1-485 of native DT, and amino acidresidues 2-133 of IL-2. DAB₄₈₅ -IL-2 is identical except that it lacksthe initial methionine residue.

DAB₃₈₉ -IL-2 consists of DAB₃₈₉ fused to amino acid residues 2-133 ofIL-2.

DAB₃₈₉ EGF consists of DAB₃₈₉ fused to EGF.

Receptor means the site to which the cell-specific polypeptide ligand(described in (d) above) binds.

Chimeric toxins of the invention display one or more of the followingadvantages: greater toxicity than that of an analagous DAB₄₈₆-containing toxin; a greater affinity for the receptor than that of ananalagous DAB₄₈₆ -containing toxin; when expressed in the cytoplasm ofE.coli, greater resistance to proteolytic degradation than thatexhibited by an analagous DAB₄₈₆ -containing toxin; greater resistanceto the inhibition of its cytotoxicity by competitive inhibitors, e.g.,the polypeptide of (d) above, than that exhibited by an analagous DAB₄₈₆-containing toxin; the ability to inhibit protein synthesis in targetcells to a given degree by a period of exposure that is shorter than theperiod of exposure required by an analogous DAB₄₈₆ -containing-toxin toinhibit protein synthesis to the same degree; or the ability to effect amore rapid onset of the inhibition of protein synthesis than that seenin an analagous DAB₄₈₆ -containing-toxin.

Aberrant expression of the epidermal growth factor receptor is acharacteristic of several malignancies including those of the breast,bladder, prostate, lung and neuroglia. Chimeric toxins of the inventionallow therapeutic targeting the cytotoxic action of diptheria toxin toEGF receptor positive tumor cells. In these chimeric toxins thesequences for the binding domain of diptheria toxin have been replacedby those for human EGF. These chimeric toxins inhibit protein synthesisby the same mechanism as diptheria toxin and are specifically cytotoxicfor human tumor cells which express elevated levels of EGF receptors.The uptake of these chimeric toxins occur with kinetics which permit useof this molecule as a powerful therapeutic agent for treatment ofmalignancies characterized by EGF receptor expression.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments and from the claims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings will first be briefly described.

Drawings

FIG. 1 is a diagram of the DT molecule and of various fusion proteins.

FIG. 2 is a depiction of the construction of the plasmids of a preferredembodiment.

FIG. 3 is a restriction map of DNA sequences encoding various chimerictoxins.

FIG. 4 is a graph of the effects of varying doses of chimeric toxins oncultured cells.

FIG. 5 is a graph of the ability of chimeric toxins to competitivelydisplace ¹²⁵ I!-labeled IL-2 from the high affinity IL-2 receptor.

FIG. 6 is the sequence of a synthetic EGF gene.

FIG. 7 is a diagramatic representation of DAB₄₈₆ EGF and DAB₃₈₉ EGF.

FIG. 8 is a graph showing the effect of EGF on DAB₄₈₆ EGF cytotoxicity.

FIG. 9 is a graph showing the effect of EGF on DAB₃₈₉ EGF cytotoxicity.

FIG. 10 is a graph showing the effect of EGF and DAB₃₈₉ EGF on the EGFbinding capacity of A431 cells.

FIG. 11 is a graph showing the ability of EGF or DAB₃₈₉ EGF to displace¹²⁵ I! EGF from EGF receptors.

FIG. 12 is a graph of the effect of length of exposure to DAB₄₈₆ EGF onthe inhibition of protein synthesis.

FIG. 13 is a graph of the effect of length of exposure to DAB₃₈₉ EGF onthe inhibition of protein synthesis.

FIG. 14 is a graph of the kinetics of the inhibition of proteinsynthesis on cells incubated with DAB₄₈₆ EGF or DAB₃₈₉ EGF.

STRUCTURE AND SYNTHESIS OF CHIMERIC TOXIN DAB₄₈₆ -IL-2

DAB₄₈₆ -IL-2 is a chimeric toxin consisting of Met followed by aminoacid residues 1 through 485 of mature DT fused to amino acid residues 2through 133 of IL-2 . The DT portion of the chimeric toxin DAB₄₈₆ -IL-2includes all of DT fragment A and the portion of DT fragment B extendingto residue 485 of mature native DT. Thus DAB₄₈₆ -IL-2 extends past thedisulfide bridge linking Cys461 with Cys471. See FIG. 1a for thestructure of DT. (The nomenclature adopted for IL-2-toxin isDAB_(486-IL-) 2, where D indicates diphtheria toxin, A and B indicatewild type sequences for these fragments, and IL-2 indicates humaninterleukin-2 sequences. Mutant alleles are indicated by a number inparentheses following DAB. The numerical subscript indicates the numberof DT-related amino acids in the fusion protein. Since the deletion ofthe tox signal sequence and expression from the trc promoter results inthe addition of a methionine residue to the N-terminus, the numbering ofDAB-IL-2 fusion toxins is +1 out of phase with that of native diphtheriatoxin.)

pDW24, which carries DAB₄₈₆ -IL-2 was constructed as follows. pUC18 (NewEngland BioLabs) was digested with PstI and BglI and the PstI-BglIfragment carrying the E.coli origin of replication, the polylinkerregion, and the 3' portion of the β-lacatamase gene (amp^(r)) wasrecovered. Plasmid pKK-233-2 (Pharmacia) was digested with PstI and BglIand the PstI-BglI fragment carrying, two transcription terminators andthe 5' portion of the β-lactamase gene was recovered. pDW22 wasconstructed by ligating these two recovered fragments together.

pDW23 was constructed by isolating a BamHI-SalI fragment encoding humanIL-2 from plasmid pDW15 (Williams et al. (1988) Nucleic Acids Res.16:10453-10467) and ligating it to BamHI/SalI digested pDW22 (describedabove).

pDW24 was constructed as follows. A BamHI-NcoI fragment carrying the trcpromoter and translational initiation codon (ATG) was isolated fromplasmid pKK233-2 (Pharmacia). The DNA sequence encoding amino acidresidues 1 through 485 of DT was obtained by digesting pABC508 (Williamset al. (1987) Protein Engineering 1:493-498) with SphI and HaeII andrecovering the HaeII-SphI fragment containing the sequence encodingamino acid residues 1 through 485 of DT. A NcoI/HaeII linker(5'CCATGGGCGC 3') was ligated to the HaeII-SphI fragment and thatcontruction was then ligated to the previously isolated BamHI-NcoIfragment carrying the trc promoter. This results in a Bam HI-SphIfragment bearing, in the following order, the trc promoter, the NcoIsite (which supplies the ATG initiator codon for Met), and the sequenceencoding residues 1 through 485 of native DT. This fragment was insertedinto pDW23 that had been digested with Bam HI and SphI. The resultingplasmid was desigated pDW24. The fusion protein (DAB₄₈₆ -Il-2) encodedby pDW24 is expressed from the trc promoter and consists of Met followedby amino acids 1 through 485 of mature DT fused to amino acids 2 through133 of human IL-2.

The sequence of DT is given in Greenfield et al. (1983) supra. Thesequence encoding IL-2 was synthesized on an Applied BiosystemsDNA-Synthesizer, as described in Williams et al. (1988) Nucleic AcidsRes. 16:10453-10467, hereby incorporated by reference. The sequence ofIL-2 is found in Williams et al. (1988) Nucleic Acids Res.16:10453-10467. Fusion of the sequence encoding mature DT to ATG usingan oligonucleotide linker is described in Bishai et al. (1987) J. Bact.169:5140-5151, hereby incorporated by reference.

pDW24 is shown in FIG. 2. The insert corresponding to DAB₄₈₆ -IL-2 isshown as a heavy line. In FIG. 2 filled circles indicate NcoI sites,open circles indicate NsiI sites, open diamonds indicate ClaI sites,filled squares indicate HpaII sites, open squares indicate SphI sites,and filled triangles indicate SalI sites.

Oligonucleotides and nucleic acids were manipulated as follows.Oligonucleotides were synthesized using cyanoethyl phosphoramiditechemistry on an Applied Biosystems 380A DNA synthesizer (AppliedBiosystems Inc., Foster City, Calif.). Following synthesis,oligonucleotides were purified by chromatography on OligonucleotidePurification Cartridges (Applied Biosystems Inc., Foster City, Calif.)as directed by the manufacturer. Purified oligonucleotides wereresuspended in TE buffer (10 mM Tris base, 1 mM EDTA, pH 8.0). To annealcomplementary strands, equimolar concentrations of each strand weremixed in the presence of 100 mM NaCl, heated to 90° C. for 10 min, andallowed to cool slowly to room temperature.

Plasmid DNA was purified by the alkaline lysis/cesium chloride gradientmethod of Ausebel et al. (1989) Current Protocols in Molecular Biology,John Wiley & Sons, N.Y. DNA was digested with restriction endonucleasesas recommended by the manufacturer (New England Biolabs, Beverly, Mass.and Bethesda Research Laboratories, Gaithersburg, Md.). Restrictionfragments for plasmid construction were extracted from agarose-TBE gels,ligated together (with or without oligonucleotide linkers) and used totransform E. coli using standard methods. Ausebel et al (1989) supra andManiatis et al. (1982), Molecular Cloning Laboratory Manual, Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y. Plasmid DNA sequencing wasperformed according to the dideoxy chain termination method of Sanger etal. (1987) Proc. Nat'l Acad. Sci USA 74:5463-5467, as modified by Kraftet al. (1988) Bio Techniques 6:544-547, using Sequenase (United StatesBiochemicals, Cleveland, Ohio).

Structure of Improved Diphtheria-IL-2 Chimeric Toxins

Expression and purification of chimeric toxins was as follows. AllDT-related IL-2 fusion proteins used herein were expressed in thecytoplasm of E. coli strain JM101 from the trc promoter, Amann et al.(1985), Gene 40:183-190, hereby incorporated by reference. RecombinantE. coli were grown in M9 minimal medium (Maniatis et al. (1982) supra)supplemented with 10 mg/ml casamino acids (Difco, Detroit, Mich.), 50μg/ml ampicillin, and 0.5 ng/ml thymine in 10 liter volumes in aMicrogen Fermentor (New Brunswick Scienctific, Edison, N.J.). Bacterialcultures were grown at 30° C., and sparged with air at 5 L/min. When theabsorbance (A₅₉₀ nm) of the culture reached 0.3, expression of chimerictox gene was induced by the addition of isopropyl-β-D-thiogalactopyranoside. Two hours after induction, bacteria wereharvested by centrifugation, resuspended in buffer #101 (50 mM KH₂ P0₄,10 mM EDTA, 750 mM NaCl, 0.1% Tween 20, pH 8.0), and lysed by sonication(Branson Sonifier). Whole cells and debris were removed bycentrifugation at 27,000×g, and the clarified extract was then filtersterilized and applied to an anti-diphtheria toxin immunoaffinitycolumn. Bound proteins were eluted with 4 M guanidine hydrochloride,reduced by the addition of β-mercaptoethanol to 1% and then sized byhigh pressure liquid chromatography on a 7.5×600 mm G4000PW column(TosoHass). Prior to use, fusion toxins were exhaustively dialysedagainst HEPES buffered Hank's balanced salt solution (Gibco), pH 7.4.Purified diphtheria toxin was purchased from List BiologicalLaboratories (Campbell, Calif.). For the production of the non-toxicCRM1001, C7(βtox-1001) was grown in 100 ml volumes of C-Y medium(Rappuoli et al. (1983) J. Bact. 153:1201-1210) in 2-liter Erlenmeyerflasks at 35° C. for 20 hrs with shaking (240 rpm). Bacteria wereremoved by centrifugation at 20,000×g for 15 min. CRM1001 wasprecipitated from the culture medium by the addition of NH₄ SO₄ to 70%saturation, and collected by centrifugation. Following dialysis against10 mM phosphate buffer, pH 7.2, CRM1001 was purified by ion exchangechromatography on DE-52 cellulose as previously described byPappenheimer et al. (1972), Immunochem. 9:891-906. The concentration ofall purified proteins was determined by using Pierce Protein Assayreagent (Pierce Chemical Co., Rockford, Ill.).

DAB(1001)₄₈₆ -IL-2 is a chimeric toxin identical to DAB₄₈₆ -IL-2 exceptfor the disruption of the disulfide bridge between Cys462 and Cys472 inDAB(1001)₄₈₆ -IL-2. DAB(1001)₄₈₆ -IL-2 was constructed by replacing the587 basepair (bp) ClaI-SphI restriction fragment which encodes most offragment B of DT) of plasmid pDW24 (which carries DAB_(486-IL-) 2 ) withthe analogous fragment from DNA encoding the TOX-1001 mutant allele ofDT. TOX-1001 encodes non-toxic diphtheria toxin-related protein CRM1001and has been shown to result from a single point mutation which changesCys471 to Tyr471, Rappuoli et al. (1986) In Protein CarbohydrateInteractions in Biological Systems, Academic Press, Inc., London, pp.295-296, hereby incorporated by reference. FIG. 3 depicts therestriction maps of DNA encoding DAB₄₈₆ -IL-2 and the correspondingfusion protein encoded by DAB₄₈₆ -IL-2 . (In FIG. 3 stippled boxesbetween the NsiI and HpaII restriction endonuclease sites designate thediphtheria toxin fragment B-related sequences which encode the membraneassociating domains. The amphipathic domain is encoded between the NsiIand ClaI sites, and the putative membrane spanning domains are encodedbetween the ClaI and HpaII sites. Hatched boxes indicate the relativeposition of internal in-frame deletion mutations.) The construction ofpDW26, which encodes the chimeric toxin with the Cys472 to Tyr472mutation, is shown in FIG. 2. Following ligation and transformation, theDNA sequence of the tox-1001 portion of the gene fusion DAB (1001)₄₈₆-IL-2 was determined in order to insure that the Cys471 to Tyr471mutation was recloned. E. coli (pDW26), was grown in M9 minimal media,cells were harvested, lysed and the fusion toxin, designatedDAB(1001)₄₈₆ -IL-2, was purified by immunoaffinity chromatography andHPLC.

The dose response capacity of DAB₄₈₆ -IL-2, CRM1001, and DAB(1001)₄₈₆-IL-2 to block ¹⁴ C!-leucine incorporation by high affinity IL-2receptor bearing HUT 102/6TG cells was determined. As anticipated,DAB_(486-IL-) 2 was highly toxic for these cells (IC₅₀ =4×10⁻¹⁰ M);whereas, CRM1001 was found to be non-toxic. In marked contrast toCRM1001, however, the fusion toxin which carries the Cys472 to Tyr472mutation, DAB(1001)₄₈₆ -IL-2 , was found to be as toxic for HUT 102/6TGcells as the wild type DAB₄₈₆ -IL-2. These results demonstrate that thefragment B disulfide bond is not required for biological activity of thefusion toxin.

HUT 102/6TG cytotoxicity assays were performed as follows. Cultured HUT102/6TG cells were maintained in RPMI 1640 medium (Gibco, Grand Island,N.Y.) supplemented with 10% fetal bovine serum (Cellect, GIBCO), 2 mMglutamine, and penicillin and streptomycin to 50 IU and 50 μg/ml,respectively. For cytotoxicity assays, cells were seeded in 96-wellV-bottomed plates (Linbro-Flow Laboratories, McLean, Va.) at aconcentration of 5×10⁴ per well in complete medium. Toxins, ortoxin-related materials, were added to varying concentrations (10⁻¹² Mto 10-6M) and the cultures were incubated for 18 hrs at 37° C. in a 5%CO₂ atmosphere. Following incubation, the plates were centrifuged for 5min. at 170×g and the medium removed and replaced with 200 μlleucine-free medium (MEM, Gibco) containing 1.0 μCi/ml ¹⁴ C!-leucine(New England Nuclear, Boston, Mass.). After an additional 90 min. at 37°C., the plates were centrifuged for 5 min. at 170×g, the medium wasremoved and the cells were lysed by the addition of 4 M KOH. Protein wasprecipitated by the addition of 10% trichloroacetic acid and theinsoluble material was then collected on glass fiber filters using acell harvester (Skatron, Sterling, Va.). Filters were washed, dried, andcounted according to standard methods. Cells cultured with medium aloneserved as the control. All assays were performed in quadruplicate.

Since the disulfide bond between Cys462-Cys472 was not required for thecytotoxic action of DAB₄₈₆ -IL-2, it was of interest to determine whatDT fragment B sequences were essential for the delivery of fragment A tothe cytosol. Several in-frame deletion mutations were introduced intothe fragment B encoding portion of the DAB₄₈₆ -IL-2 toxin gene, FIGS.1b, 2, and 3. FIG. 1b shows the structure of DAB₄₈₆ -IL-2 and variousmutants derived from DAB₄₈₆ -IL-2. In FIG. 1b a wide bar indicates thefusion protein, narrow connecting lines represent deletions, numbersabove the bars are amino acid residue numbers in the DAB nomenclature,numbers below the bars correspond to the amino acid residue numbering ofnative DT, cross hatching indicated amphipathic regions, darkened areascorrespond to the transmembrane region, IL-2-2-133 indicates amino acidresidues 2-133 of IL-2 , Ala=alanine, Asn=asparagine, Asp=aspartic acid,Cys=cysteine, Gly=glycine, His=histidine, Ile=isoleucine,Met=methionine, Thr=threonine, Tyr=tyrosine, and Val=valine.

The first mutant, DAB₃₈₉ -IL-2 was constructed by removing a 309 bpHpaII-SphI restriction fragment from pDW24 and replacing it witholigonucleotide linker 261/274 (Table 1) to generate plasmid pDW27 (FIG.1). This linker restores fragment B sequences from Pro383 to Thr387, andallows for in-frame fusion to IL-2 sequences at this position. Thus, inDAB₃₈₉ -IL-2 the 97 amino acids between Thr387 and His485 have beendeleted.

                                      TABLE 1    __________________________________________________________________________    Oligonucleotide linkers             oligonucleotide    construct             identification number                        linker    __________________________________________________________________________    DAB.sub.389 -IL-2             274        5'-CG GGT CAC AAA ACG CAT G-3'             261              CCA GTG TTT TGC                        1/2 HpaII          1/2 SphI    DAB.sub.295 -IL-2             292        5'-C GAT GGT GTG CAT G-3'             293              TA CCA CAC                        1/2 ClaI        1/2 SphI    DAB(Δ205-             337        5'-TA AAT AT-3'    289).sub.486 -IL-2             338        ACG TAT TTA TAG C                        1/2 NsiI        1/2 ClaI    DAB(Δ205-             337        5'-TA AAT AT-3'    289).sub.486 -IL-2             338        ACG TAT TTA TAG C                        1/2 NsiI        1/2 ClaI    __________________________________________________________________________

In a similar fashion, a 191 amino acid in-frame deletion was constructedby removing a ClaI-SphI restriction fragment from pDW24 and replacing itwith the 292/293 oligonucleotide linker (Table 1) to form plasmid pDW28which encodes DAB₂₉₅ -IL-2. In this case, the in-frame deletionencompasses the putative membrane-spanning helices that have beenpredicted by Lambotte et al. (1980) J. Cell. Biol. 87:837-840, to play arole in the delivery of fragment A to the eukaryotic cell cytosol.

Purified, DAB₃₈₉ -IL-2 and DAB₂₉₅ -IL-2 were found to haveelectrophoretic mobilities of 57 kDa and 47 kDa, respectively. The doseresponse analysis on HUT 102/6TG cells is shown in FIG. 4. In FIG. 4DAB₄₈₆ -IL-2 is indicated by filled squares; DAB₃₈₉ -IL-2 is indicatedby filled circles; DAB₂₉₅ -IL2 is indicated by open circles;DAB(Δ205-289) ₄₈₆ -IL-2 (see below) is indicated by open squares; andDAB(Δ205-289)₃₈₉ -IL-2 (see below) is indicated by open triangles.DAB₄₈₆ -IL-2 and DAB₃₈₉ -IL-2 exhibited an IC₅₀ of approximately 4×10⁻¹⁰M and 1×10⁻¹⁰ M, respectively. In marked contrast, the IC₅₀ of DAB₂₉₅-IL-2 was approximately 1,000-fold lower (4×10⁻⁷ M). These resultssuggest that fragment B sequences between Thr387 and His486 do not playa major role in the delivery of fragment A to the cytosol. Sequencesbetween Ser292 and Thr387 on the other hand are essential for theefficient delivery of fragment A.

Surprisingly, DAB₃₈₉ -IL-2 possessed much greater activity than didDAB₄₈₆ -IL-2. DAB₃₈₉ -IL-2, which lacks native DT residues 387 through483, and which has increased toxic activity, leaves the hydrophobictransmembrane segment located approximately between native DT residues346 and 371 intact. See Lambotte et al. (1980) J. Cell Biol. 87:837-840,hereby incorporated by reference, for a characterization of thetransmembrane region. DAB₂₉₅ -IL-2, which removes native DT residues 291through 481, and which has greatly reduced toxicity, removes thetransmembrane region (346-371).

In order to rule out the possibility that the reason for the low potencyof DAB₂₉₅ -IL-2 for HUT 102/6TG cells was related to altered binding tothe high affinity IL-2 receptor, we have conducted a series ofcompetitive displacement experiments using ¹²⁵ I!-rIL-2 . FIG. 5 showsthe competitive displacement of ¹²⁵ I!-labeled IL-2 from the highaffinity IL-2 receptor by unlabeled rIL-2 depicted by filled circles;DAB₄₈₆ -IL-2 depicted by open triangles; DAB₃₈₉ -IL-2 depicted by closedsquares; DAB₂₉₅ -IL-2 depicted by closed triangles; DAB(Δ205-289)₄₈₆-IL-2 (see below) depicted by open circles; and DAB(Δ205-289)₃₈₉ -IL-2(see below) depicted by open squares. The concentration of ¹²⁵ !-IL-2used was 10 pM and the specific activity was approximately 0.7 μCi/pmol.As shown in Table 2, both DAB₃₈₉ -IL-2 and DAB₂₉₅ -IL-2 were found tohave an apparent K_(d) that is approximately 3-times lower than that ofDAB₄₈₆ -IL-2 (K_(d) =8×10⁻⁹ M vs. K_(d) =2.5×10⁻⁸ M). It is particularlysignificant that competitive displacement experiments showed that bothDAB₃₈₉ -IL-2 and DAB₂₉₅ -IL-2 bind more avidly to the high affinity IL-2receptor than does DAB₄₈₆ -IL-2 (Kd=8×10⁻⁹ and 8.4×10⁻⁹ M vs.Kd=2.5×10⁻⁸ M). These results provide evidence that fusion of IL-2sequences to toxophores of smaller mass may serve to position the IL-2binding domain for more favorable receptor interaction.

It is of interest to note that while DAB₂₉₅ -IL-2 binds more avidly tothe high affinity IL-2 receptor than DAB₄₈₆ -IL-2, its cytotoxicactivity is at least 1,000-fold lower (FIG. 4). These results indicatedthat avid binding to the target receptor is not in itself sufficient forthe biologic activity of the DT-related IL-2 fusion toxins, and thatfragment B sequences between Ser292 and Thr387 are essential for apost-receptor binding event in the intoxication process.

                  TABLE 2    ______________________________________    Relative ability of rIL-2 and DAB-IL-2 related fusion    proteins to displace  .sup.125 I!--rIL-2 from high    affinity IL-2 receptors on HUT 102/6TG cells    unlableled ligand                    apparent K.sub.d                              K.sub.d DAB-IL-2/rIL-2    ______________________________________    rIL-2           .sup. 1.7 × 10.sup.-10                              --    DAB.sub.486 -IL-2                    2.5 × 10.sup.8                              147    DAB.sub.389 -IL-2                    8.0 × 10.sup.9                               47    DAB.sub.295 -IL-2                    8.4 × 10.sup.9                               49    DAB(Δ205-289).sub.486 -IL-2                    1.0 × 10.sup.-7                              588    DAB(Δ205-289).sub.389 -IL-2                    2.9 × 10.sup.-8                              170    ______________________________________

Competitive displacement of ¹²⁵ I!-rIL-2 by rIL-2 and DAB-IL-2 fusiontoxins was determined as follows. The radiolabeled IL-2 binding assaywas performed essentially as described by Wang et al. (1987) J. Exp.Med. 166:1055-1069. Cells were harvested and washed with cell culturemedium. HUT 102/6TG cells were resuspended to 5×10⁶ per ml and incubatedwith ¹²⁵ I!-rIL-2 (0.7 μCi/pmol) in the presence or absence ofincreasing concentrations of unlabeled rIL-2 or the DAB-IL-2 fusiontoxins for 30 min. at 37° C. under 5% CO₂. The reaction was thenoverlayed on a mixture of 80% 550 fluid (Accumetric Inc., Elizabethtown,Ky.): 20% parafin oil (d=1.03 g/ml) and microcentrifuged. The aqueousphase and the pellet of each sample, representing free and bound ligand,respectively, was then counted in a Nuclear Chicago gamma counter.Apparent dissociation constants, K_(d), were determined from theconcentrations of unlabeled ligand required to displace 50% ofradiolabeled rIL-2 binding to receptors.

In order to test the hypothesis that an amphipathic region (amino acids210-252 in DAB_(486-IL-) 2) plays a role in the intoxication process,in-frame deletions of the 85 amino acid encoding region from NsiI toClaI of both pDW24 and pDW27 to form pDW30 (containing DAB(Δ205-289)₄₈₆-IL-2) and pDW31 (containing DAB(Δ205-289)₃₈₉ -IL-2 ), respectively(FIGS. 2 and 3; Table 1) were constructed. Following ligation andtransformation, the DAB-IL-2 related fusion proteins were expressed andpurified, as described above. As shown in FIG. 4, the deletion offragment B sequences which include the amphipathic region result in amarked loss of cytotoxic activity against high affinity IL-2 receptorpositive cells in vitro. It is of interest to note that DAB(Δ205-289)₃₈₉ - IL-2 was found to displace radiolabeled IL-2 from thehigh affinity receptor almost as well as DAB₄₈₆ -IL-2; whereas,DAB(Δ205-289)₄₈₆ -IL-2 was found to bind 4-fold less avidly to thereceptor (FIG. 5).

Increased Resistance to Proteolytic Degradation

The chimeric toxin encoded by DAB₃₈₉ -IL-2 is more resistant toproteolytic degradation than is the chimeric toxin encoded by DAB₄₈₆-IL-2. When purified, as described above, and analysed onSDS-polyacrylamide gels, the DAB₃₈₉ -IL-2 hybrid toxin is accompanied byvery few degradation products (as evidenced by the relative absence ofbands of smaller size than that of the intact chimeric toxin). PurifiedDAB₄₈₆ -IL-2 on the other hand is accompanied by numerous dark bands oflower molecular weight than the intact chimeric toxin. These lowermolecular weight bands react with anti-DAB₄₈₆ -IL-2 antibodies,supporting the conclusion that they are degradation products.

Sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE)was performed according to the method of Laemmli (1970) Nature227:680-685 using 12% gels and a Mini-Protein II gel apparatus (BioRad).Proteins were fixed in 12.5% trichloroacetic acid for 5 min and stainedwith Coomassie brilliant blue according to the Diezal procedure, Diezalet al. (1972) Anal. Biochem. 48:617-624.

Construction of Fusion Genes Encoding DT-EGF Chimeric Toxins

DAB₄₈₆ EGF and DAB₃₈₉ EGF can be constructed in a manner analogous tothat in which DAB₄₈₆ -IL-2 and DAB₃₈₉ -IL-2 were constructed, by methodsknown to those skilled in the art. To construct a plasmid encodingDAB₄₈₆ fused to EGF, plasmid pDW24 (which encodes DAB₄₈₆ fused to IL-2)is digested with SphI and HindIII to remove the IL-2 coding sequence.The resulting pDW24 SphI-HindIII fragment containing the sequenceencoding DT residues 1-485 is ligated to a synthetic SphI-HindIIIfragment encoding EGF to yield a plasmid encoding DAB₄₈₆ fused to EGF.The EGF fragment, shown in FIG. 6, was synthesized, as described, usingpreferred codons for expression in E.coli (see Grosjean et al. (1982)Gene 18:199-209, hearby incorporated by reference). The syntheticfragment includes appropriate linkers at the 5' and 3' ends forinsertion into the plasmid and for in-frame fusion to the DT codingsequence.

To construct a plasmid encoding DAB₃₈₉ fused to EGF a similar protocolcan be followed, except that pDW27 (which encodes DAB₃₈₉ fused to IL-2)is used in place of pDW24. The IL-2 encoding DNA is removed from pDW27by digestion with SphI and HindIII and EGF encoding DNA is inserted inits place, resulting in a DAB₃₈₉ fused in frame to EGF. The samesynthetic EGF fragment used in the construction of the DAB₄₈₉ EGF fusion(FIG. 6) can be used.

Those skilled in the art will realize that the protocols given above arenot the only way of making the chimeric toxins of the invention.Refinements include changes in pDW24, pDW27, and plasmids derivedtherefrom directed toward compliance with the Good ManufacturingPractises of the Food and Drug Administration, e.g., the inclusion ofthe lacI^(q) gene (Amersham) and the replacement of the ampicillinresistance gene (amp^(r)) with the gene for neomycin/kanamycinresistance from Tn5 (Pharmacia) in the plasmids that are used forexpression of the chimeric toxins of the invention. These alterationscan be performed without undue experimentation by one skilled in theart.

The Biological Activity of DT-EGF Chimeric Toxins

DAB₄₈₆ EGF and DAB₃₈₉ EGF are the products of fusion genes in which thereceptor binding domain of DT has been removed and replaced with DNAencoding human EGF. As shown in FIG. 7, the resulting proteins containthe enzymatically active fragment A of DT and the lipid associatingdomains of fragment B of DT required for translocation of fragment Ainto the cytosol. DAB₃₈₉ EGF differs from DAB₄₈₆ EGF by the deletion ofthe 97 amino acids immediately 5' to amino acid residue 484 of DT. TheEGF portion of both DAB₄₈₆ EGF and DAB₃₈₉ EGF governs receptor binding.Thus, these molecules have the potential to specifically target thecytotoxicity of DT to tumor cells characterized by EGF receptorexpression.

DT-EGF Chimeric Toxins Are Toxic to EGF-Receptor-Bearing Cells.

The cytotoxicity of DAB₄₈₆ EGF for a panel of human cell lines wasassessed and compared to A431 cells (ATCC CRL 1555), a human epidermoidcarcinoma cell line with a high number of EGF receptors. The results areshown in Table 3. Included in the study were human tumor cell lineswhich have been reported to express high numbers of EGF receptors (e.g.,BT-20, HeLa, LNCaP and U-87 MG) as well as human tumor cell lines (e.g.,C91/PL, BeWo and A375) and normal tissue cell lines (e.g., WI-38, Hs 67and HEPM) expressing few or no EGF receptors. Cytotoxicity was evaluatedas follows. Cells were plated in triplicate wells of 96 well plates withDAB₄₈₆ EGF in assay medium appropriate to the cell type (see Table 3).DAB₄₈₆ EGF concentrations were between 10⁻¹⁵ and 10⁻⁷ M. Following a20-hour incubation, cells were labeled with ¹⁴ C! leucine, trypsinized,harvested onto glass fiber filter mats and counted to determine thepercent of control incorporation. Cell lines exhibiting an IC₅₀ forDAB₄₈₆ EGF of less than 0.5 nM were considered sensitive.

                  TABLE 3    ______________________________________    The effect of a DT-EGF chimeric toxin on various cell lines    ______________________________________    Tumor Cell lines    Cell Line   Tissue/Type      Sensitivity    ______________________________________    A431        vulval epidermoid carcinoma                                 +    A549        lung carcinoma   +    KB          oral epidermoid carcinoma                                 +    BT-20       breast adenocarcinoma                                 +    HeLa S3     cervical carcinoma                                 +    T47D        breast ductal carcinoma                                 +    LNCaP.FG    prostate carcinoma                                 +    HOS         osteosarcoma     +    U-87 MG     glioblastoma/astrocytoma                                 +    C91/PL      HTLV-1 transformed T cell                                 -    BeWo        choriocarcinoma  -    A375        malignant melanoma                                 -    MCF-7       breast adenocarcinoma                                 -    SNU-C2B     cecum carcinoma  -    ______________________________________    Normal Cell Lines    Cell Line   Tissue           Sensitivity    ______________________________________    WI-38       diploid lung fibroblast                                 -    Hs 67       thymus           -    CCD-18Co    colon fibroblast -    HISM        smooth muscle, jejunum                                 -    FH74s Int   fetal small intestine                                 -    HEPM        embryonic palatal                                 -                mesenchyme    ______________________________________

Growth conditions and passage schedules used were those defined by ATCC(except as noted below). Culture media were as follows: A431 (ATCCCRL1555), DMEM+10% FCS; A549 (ATCC CCL185) Ham's F12+10% FCS; KB (ATCCCCL17), DMEM+NEAA+10% FCS; BT-20 (ATCC HTB19), MEM+10% FCS; HeLa S3(ATCC CCL2.2), Ham's F12+10% FCS; T47D (ATCC HTB133), RPMI 1640+10% FCS;LNCaP.FG (ATCC CRL1740), RPMI 1640+10% FCS; HOS (ATCC CRL1543), MEM+10%FCS; U-87 MG (ATCC HTB14), MEM+10% FCS; C91/PL (from Robert Swartz,NEMC, Boston, Mass., see Bacha et al. (1988) J. Exp. Med. 167:612 forgrowth conditions), RPMl 1640+15% FCS; BeWo (ATTC CCL98), Ham's F12+15%FCS; A375 (ATCC CRL 1619), DMEM+10% FCS; MCF-7 (ATCC TB22) MEM+10% FCS;SNU-C2B (ATCC CCL250) RPMl 1640+10% FCS; WI-38 (ATCC CCL75), Eagle'sBasal+10% FCS; Hs 67 (ATCC HTB 163), DMEM+10% FCS; CCD-18Co (ATCC CRL1459), MEM+10% FCS; HISM (ATCC CRL 1692), DMEM+10% FCS; FHs74Int (ATCCCCL241), DMEM+10% FCS; HEPM (ATCC CRL 1486), MEM+10% FCS.DMEM=Dulbecco's modified Eagles Medium; MEM=Minimum Essential Medium;NEAA=Non-Essential Amino Acids; FCS=Fetal Calf Serum; ATCC=American TypeCulture Collection.

To demonstrate that the cytotoxic action of DAB₄₈₆ EGF and DAB₃₈₉ EGFare mediated selectively by the EGF receptor, A431 cells were plated intriplicate wells of 96 well plates with DAB₄₈₆ EGF (FIG. 8) or DAB₃₈₉EGF (FIG. 9) in the presence of the specific competitor of the EGFreceptor, human EGF (Upstate Biotechnologies, Inc.) (10⁻⁷ M), in assaymedium (DMEM+10% FCS). In FIG. 8 solid squares indicate DAB₄₈₆ EGF andsolid triangles indicate DAB₄₈₆ EGF+EGF. In FIG.9 solid squares indicateDAB₃₈₉ EGF and solid triangles indicate DAB₃₈₉ EGF+EGF. Following a20-hour incubation at 37° C., cells were labeled with ¹⁴ C! leucine,trypsinized, harvested onto glass fiber filter mats and counted todetermine the percent of control incorporation. The results show that,in the absence of EGF, DAB₄₈₆ EGF and DAB₃₈₉ EGF inhibit proteinsynthesis with an IC₅₀ of 3×10⁻¹² M and 3×10⁻¹³ M, respectively. EGFalmost completely abolishes this activity. Likewise, anti-EGF(Biomedical Technologies, Inc.) and anti-EGF receptor (UpstateBiotechnologies, Inc.) also abolish the cytotoxicity of DAB₄₈₆ EGF andDAB₃₈₉ EGF while the nonspecific competitors, transferrin (Sigma)anti-transferrin (Dako), and anti-transferrin receptor (Dako), have noeffect. These results demonstrate that DAB₄₈₆ EGF and DAB₃₈₉ EGF arepotent and specific cytotoxic agents. Note that DAB₃₈₉ EGF isapproximately 10 times more potent than DAB₄₈₆ EGF.

DAB₃₈₉ EGF, like EGF, induces down regulation of the EGF receptor,providing further evidence for the EGF receptor-specific nature ofDT-EGF chimeric toxins. Binding and internalization of EGF induces downregulation of the EGF receptor which can be detected as a decrease in¹²⁵ I!EGF binding capacity (Krupp et al. (1982) J. Biol. Chem.257:11489). The ability of DAB₃₈₉ EGF to induce EGF receptorinternalization and subsequent down regulation was evaluated andcompared to that induced by native EGF. The results are shown in FIG.10. In FIG. 10 open squares indicate EGF and closed diamonds indicateDAB₃₈₉ EGF. A431 cells in triplicate wells of 24 well plates werepreincubated with EGF or DAB₃₈₉ EGF (10⁻⁸ M) for the indicated times inDMEM+0.1% BSA (bovine serum albumin) at 37° C. The cells were thenplaced on ice and acid stripped (with 0.2 M acetic acid, 0.5 M NaCl) toremove bound, but not internalized, EGF or DAB₃₈₉ EGF. EGF bindingcapacity was measured by incubating the cells, on ice, with ¹²⁵ I !EGF.Following a 90-minute incubation the cells were washed, solubilized, andcounted.

An EGF receptor-dependent interaction is also shown by the fact thatDAB₃₈₉ EGF, like EGF, displaces ¹²⁵ I!EGF from the EGF receptor, asshown in FIG. 11. In FIG. 11 open squares indicate EGF and soliddiamonds indicate DAB₃₈₉ EGF. Results in FIG. 11 are expressed as apercent of control (no competition) cpm. The ability of DAB₃₈₉ EGF todisplace high affinity ¹²⁵ I!EGF binding to A431 cells was evaluated asfollows. A431 cells, plated in triplicate wells of 24 well plates, werepreincubated in binding media (phosphate buffered saline pH 7.2+0.1%BSA+15 mM sodium azide+50 mM 2-deoxyglucose) for 1 hour at 37° C. andthen incubated with ¹²⁵ I!EGF in binding media in the presence of DAB₃₈₉EGF or EGF. Following incubation, the cells were washed, solubilized andcounted. The results are summarized in Table 4.

In Table 4 EC₅₀ is the concentration resulting in displacement of 50% ofthe ¹²⁵ I!EGF.

                  TABLE 4    ______________________________________    Displacement of  .sup.125 I! EGF Binding by EGF and DAB.sub.389 EGF                            fold over                                     fold over    Competition              EC.sub.50      .sup.125 I! EGF                                     EGF    ______________________________________    EGF       1.0 × 10.sup.-8 M                             20      --    DAB.sub.389 EGF              4.5 × 10.sup.-7 M                            900      45    ______________________________________

Cytotoxicity of DT-EGF Chimeric Toxins is DT Dependent.

Upon binding to its receptor, the cellular uptake of native DT occurs byendocytosis of clathrin coated vesicles (Middlebrook et al. (1978) J.Biol. Chem. 253:7325). DT is then found in endosomes where the low pHinduces a conformational change facilitating the translocation of theenzymatic fragment A portion of DT into the cytosol. To determine if thecytotoxicity of DAB₄₈₆ EGF and DAB₃₈₉ EGF is also dependent upon thesame pathway, A431 cells were plated in sextuplicate wells of 96 wellplates containing DAB₄₈₆ EGF, DAB₃₈₉ EGF or DMEM+10% FCS in the absenceor presence of chloroquine (10⁻⁵ M) (Sigma). Chloroquine is alysosomotropic compound which prevents acidification of endosomes (Kimet al. (1965) J. Bacteriol. 90:1552). Following a 20-hour incubation at37° C., the cells were labeled with ³ H! leucine, trypsinized, harvestedonto glass fiber filter mats and counted. The results are shown in Table5, expressed as the percent of control (no DAB₄₈₆ EGF or DAB₃₈₉ EGF)incorporation and represent the mean of three experiments. The resultsshow that chloroquine blocks the cytotoxicity of DT-EGF chimeric toxins.

                  TABLE 5    ______________________________________    Sensitivity of DAB-EGF Chimeric Toxin-Cytotoxicity to    Chloroquine    Percent of Control Incorporation                   No Addition                           Chloroquine    ______________________________________    DAB.sub.486 EGF Concentration    0                100        86    10.sup.-8 M       5        60    10.sup.-9 M      25        96    DAB.sub.389 EGF Concentration    0                100       73    10.sup.-11 M     4         61    10.sup.-12 M     57        100    ______________________________________

Following translocation into the cytosol, fragment A catalyzes thecleavage of NAD and the covalent linkage of ADP-ribose to elongationfactor 2 (EF-2) resulting in the inhibition of protein synthesis (Bachaet al. (1983) J. Biol. Chem. 258:1565). To evaluate the mechanism bywhich DAB₄₈₆ EGF inhibits protein synthesis, A431 cells were plated intriplicate wells of 24 well plates containing DT, DAB₄₈₆ EGF, orcomplete medium. Following a 20-hour incubation at 37° C., the cellswere washed and incubated in lysis buffer (10 mM Tris, 10 mM NaCl, 3 mMMg Cl₂, 10 mM thymidine, 1 mM EGTA, 1% TRITON X-100) with ³² P!NAD inthe presence of purified DT fragment A (Calbiochem). TCA precipitableextracts were collected on glass fiber filters and counted to quantitatethe percent of control EF-2 available for ADP-ribosylation. The resultsof these experiments are shown in Table 6. DAB₄₈₆ EGF, like DT, reduced(in a dosage dependent manner) the amount of EF-2 available for ADPribosylation.

                  TABLE 6    ______________________________________    ADP-Ribosylation of EF-2 by DAB.sub.486 EGF                           Percent of Control Level                           of EF-2 Available for    Toxin       Concentration                           for ADP-ribosylation    ______________________________________    DT          10.sup.-8 M                           <1                10.sup.-9 M                           17    DAB.sub.486 EGF                10.sup.-8 M                           13                10.sup.-9 M                           20    ______________________________________

DAB₃₈₉ EGF Is An Improved Chimeric Toxin.

DAB₃₈₉ EGF is far more toxic than is DAB₄₈₆ EGF. As shown in FIGS. 8 and9, DAB₃₈₉ EGF exhibits an IC₅₀ for the inhibition of protein synthesisin A431 cells approximately 10 times lower than that of DAB₄₈₆ EGF(DAB₃₈₉ EGF IC₅₀ =3×10⁻¹³ M; DAB₄₈₆ EGF IC₅₀ =3×10⁻¹² M).

The greater potency of DAB₃₈₉ EGF is also shown in experiments measuringthe rapidity with which DAB₃₈₉ EGF and DAB₄₈₆ EGF kill A431 cells. FIGS.12 and 12 show the exposure time (of A431 cells to DAB₄₈₆ EGF or DAB₃₈₉EGF) required to induce maximal inhibition of protein synthesis. Cellswere exposed to DAB₄₈₆ EGF (5×10⁻⁹ M) (FIG. 12) or DAB₃₈₉ EGF (5×10⁻⁹ M)(FIG. 13) for the indicated times and then washed of unbound DAB₄₈₆ EGFor DAB₃₈₉ EGF. Following an overnight incubation in complete media(DMEM+10% FCS), the cells were labeled with ¹⁴ C! leucine. The resultsshow that near maximal inhibition of protein synthesis occurs followinga 15-minute exposure to DAB₃₈₉ EGF while a greater than 75-minuteexposure is required for DAB₄₈₆ EGF.

The kinetics of protein synthesis inhibition in DAB₃₈₉ EGF or DAB₄₈₆ EGFtreated A431 cells is shown in FIG. 14. To examine the kinetics ofprotein synthesis inhibition A431 cells were incubated with DAB₄₈₆ EGF(5×10⁻⁹) or DAB₃₈₉ EGF (5×10⁻⁹ M) in complete medium at 37° C. At theindicated times, DAB₄₈₆ EGF or DAB₃₈₉ EGF was removed and the cells werelabeled with ¹⁴ C! leucine for 1 hour. The results indicate that thereis a 50% reduction in protein synthesis following a 1-hour incubationwith DAB₃₈₉ EGF while maximal inhibition occurs by 4 hours. Maximalinhibition of protein synthesis occurs more than 6 hours followingincubation with DAB₄₈₆ EGF.

Use

The improved chimeric toxins of the invention are administered to amammal, e.g., a human, suffering from a medical disorder, e.g., cancer,or other conditions characterized by the presence of a class of unwantedcells to which a polypeptide ligand can selectively bind. The amount ofprotein administered will vary with the type of disease, extensivenessof the disease, and size of species of the mammal suffering from thedisease. Generally, amounts will be in the range of those used for othercytotoxic agents used in the treatment of cancer, although in certaininstances lower amounts will be needed because of the specificity andincreased toxicity of the improved chimeric toxins.

The improved chimeric toxins can be admnistered using any conventionalmethod; e.g., via injection, or via a timed-release implant. In the caseof MSH improved chimeric toxins, topical creams can be used to killprimary cancer cells, and injections or implants can be used to killmetastatic cells. The improved chimeric toxins can be combined with anynon-toxic, pharmaceutically-acceptable carrier substance.

Other Embodiments

Other embodiments are within the following claims. For example, chimerictoxins have been constructed, by methods known to those skilled in theart, in which DAB₃₈₉ and DAB₄₈₆ have been fused to interleukin 4 (IL-4).DAB₃₈₉ -IL-4 is about 10 times more cytotoxic than is DAB₄₈₆ -IL-4.DAB₃₈₉ has also been fused to interleukin 6. DAB₄₈₆ and DAB₃₈₉ have alsobeen fused to human chorionic gonadotropin. The improved chimeric toxinsof the invention include portions of DT fused to any cell-specificpolypeptide ligand which has a binding domain specific for theparticular class of cells which are to be intoxicated. Polypeptidehormones are useful such ligands. Chimeric toxins, e.g., those madeusing the binding domain of α or β MSH, can selectively bind tomelanocytes, allowing the construction of improved DT-MSH chimerictoxins useful in the treatment of melanoma. Other specific-bindingligands which can be used include insulin, somatostatin, interleukins Iand III, and granulocyte colony stimulating factor. Other usefulpolypeptide ligands having cell-specific binding domains are folliclestimulating hormone (specific for ovarian cells), luteinizing hormone(specific for ovarian cells), thyroid stimulating hormone (specific forthyroid cells), vasopressin (specific for uterine cells, as well asbladder and intestinal cells), prolactin (specific for breast cells),and growth hormone (specific for certain bone cells). Improved chimerictoxins using these ligands are useful in treating cancers or otherdiseases of the cell type to which there is specific binding.

For a number of cell-specific ligands, the region within each suchligand in which the binding domain is located is now known. Furthermore,recent advances in solid phase polypeptide synthesis enable thoseskilled in this technology to determine the binding domain ofpractically any such ligand, by synthesizing various fragments of theligand and testing them for the ability to bind to the class of cells tobe labeled. Thus, the chimeric toxins of the invention need not includean entire ligand, but rather may include only a fragment of a ligandwhich exhibits the desired cell-binding capacity. Likewise, analogs ofthe ligand or its cell-binding region having minor sequence variationsmay be synthesized, tested for their ability to bind to cells, andincorporated into the hybrid molecules of the invention. Other potentialligands include monoclonal antibodies or antigen-binding, single-chainanalogs of monoclonal antibodies, where the antigen is a receptor orother moiety expressed on the surface of the target cell membrane.

What is claimed is:
 1. A DNA molecule encoding a chimeric toxin whichbinds selectively to a predetermined class of cells comprising proteinfragments joined together by peptide bonds, said chimeric toxincomprising, sequentially from N-terminus to C-terminus,(a) a firstfragment comprising Fragment A of native diphtheria toxin; (b) a secondfragment comprising a portion of Fragment B of native diphtheria toxinwhich together with said first Fragment A forms the 1₁ domain of nativediphtheria toxin, said portion also comprising the hydrophobictransmembrane domain which is amino acids 346-371, said portionexcluding the 1₂ domain which is amino acids 461-471, the eukaryoticbinding domain which is amino acids 485-535 of native diphtheria toxin,and at least 50 amino acids N-terminal to said eukaryotic bindingdomain; and (c) a third fragment comprising at least a portion of thebinding domain of a cell-specific polypeptide ligand effective to causesaid chimeric toxin to bind selectively to the predetermined class ofcells which bear a receptor to said ligand.
 2. The DNA molecule of claim1, wherein said second fragment excludes at least 80 amino acidsN-terminal to said eukaryotic binding domain.
 3. The DNA molecule ofclaim 1, wherein said second fragment is amino acid residues 194-371 ofnative diphtheria toxin.
 4. The DNA molecule of claim 1, wherein saidthird fragment comprises a portion of the binding domain of EGFeffective to cause said chimeric toxin to bind selectively to thepredetermined class of cells which bear a receptor to EGF.
 5. The DNAmolecule of claim 1, wherein said third fragment comprises EGF.
 6. TheDNA molecule of claim 1, wherein said third fragment comprises at leasta portion of the binding domain of IL-2 effective to cause said chimerictoxin to bind selectively to the predetermined class of cells which beara receptor to IL-2.
 7. The DNA molecule of claim 1, wherein said thirdfragment comprises amino acids 2 to 133 of human IL-2.
 8. The DNAmolecule of claim 1, wherein said third fragment comprises at least aportion of the binding domain of IL-4 effective to cause said chimerictoxin to bind selectively to the predetermined class of cells which beara receptor to IL-4.
 9. The DNA molecule of claim 1, wherein said thirdfragment comprises IL-4.
 10. The DNA molecule of claim 1, wherein thirdfragment comprises at least a portion of the binding domain of IL-6effective to cause said chimeric toxin to bind selectively to thepredetermined class of cells which bear a receptor to IL-6.
 11. The DNAmolecule of claim 1, wherein said third fragment comprises IL-6.
 12. TheDNA molecule of claim 1, wherein said chimeric toxin possesses any ofgreater toxicity than that of a toxin comprised of DAB₄₈₆ fused to saidthird fragment, a greater affinity for the receptor on cells of saidpredetermined class to which said chimeric toxin binds than that of atoxin comprised of DAB₄₈₆ fused to said third fragment, greaterresistance to proteolytic degradation than that exhibited by a toxincomprised of DAB₄₈₆ fused to said third fragment, the ability to inhibitprotein synthesis to a given degree by a period of exposure that isshorter than the period of exposure required by DAB₄₈₉ fused to saidthird fragment to inhibit protein synthesis to the same degree, or theability to effect a more rapid onset of the inhibition of proteinsynthesis than that exhibited by DAB₄₈₆ fused to said third fragment.13. The DNA molecule of claim 1, wherein said chimeric toxin is at leastabout four times as cytotoxic to said predetermined class of cells thanDAB₄₈₆ fused to said third fragment.
 14. The DNA molecule of claim 1,wherein said chimeric toxin which is at least about ten times ascytotoxic to said predetermined class of cells than DAB₄₈₆ fused to saidthird fragment.
 15. A recombinant vector containing the DNA molecule ofclaim
 1. 16. A host cell comprising the vector of claim
 15. 17. A methodof producing a chimeric toxin which binds selectively to a predeterminedclass of cells comprising protein fragments joined together by peptidebonds, said chimeric toxin comprising, sequentially from N-terminus toC-terminus,(a) a first fragment comprising Fragment A of nativediphtheria toxin; (b) a second fragment comprising a portion of FragmentB of native diphtheria toxin which together with said first Fragment Aforms the 1₁ domain of native diphtheria toxin, said portion alsocomprising the hydrophobic transmembrane domain which is amino acids346-371, said portion excluding the 1₂ domain which is amino acids461-471, the eukaryotic binding domain which is amino acids 485-535 ofnative diphtheria toxin, and at least 50 amino acids N-terminal to saideukaryotic binding domain; and (c) a third fragment comprising at leasta portion of the binding domain of a cell-specific polypeptide ligandeffective to cause said chimeric toxin to bind selectively to thepredetermined class of cells which bear a receptor to said ligand saidmethod comprising culturing the host cell of claim 16 and recovering thechimeric toxin therefrom.